Our long-term goals are to identify the genetic basis for disease traits in the lysosomal storage disorder Niemann-Pick Type C1 (NPC1), to study NPC1 disease pathogenesis, and to develop diagnostic and treatment paradigms for this disease. NPC1 is an autosomal recessive, neurovisceral lipid storage disorder that presents with variable hepatosplenomegaly, vertical supranuclear ophthalmoplegia, progressive ataxia, dystonia, and dementia. Our group has a long-term commitment to studying several aspects of this disease including those involving genetic diagnostic, prognostic and therapeutic approaches. The approaches we develop for assessing and treating NPC1 disease will also be used as a model for other rare human diseases. 1. Prognostics: The emergence of precision medicine brings the potential that individual variations of genomic sequence will allow prognosis of disease progression and direct decisions regarding appropriate therapeutic interventions. Earlier disease prediction is being facilitated by the identification of disease-causing mutations at younger ages, increasing the efficacy of genomic-directed treatment. However in many diseases such as NPC1, there is little concordance between the predicted functional consequences of identified coding mutations and clinically relevant parameters such as the time of onset or the severity of the disease. In these instances, new assays with predictive capacity need to be developed to allow improved care and treatment. We previously developed such an assay, whereby we can potentially predict the time of onset of neurological symptoms in NPC1 disease by measuring the severity of lysosomal defects in fibroblasts from NPC1 patients using a commercial LysoTracker stain. Over the last year we published two new reports identifying several new fluorescent probes (LysoProbes I-VI) that facilitate lysosomal pH monitoring and characterization of lysosome-dependent apoptosis. LysoProbes are superior to the LysoTracker marker since the fluorescent signals are both stable and highly selective, and they will aid in characterization of lysosome morphology and trafficking. We have utilized this assay to undertake whole genome siRNA screens to identify modifier loci that contribute to NPC1 genetic variation and to identify potential treatment paradigms for testing in vitro and in vivo. We have also published a study where we generated an induced pluripotent stem cell line from a subject homozygous for the most frequent NPC1 mutation (p.I1061T) and subsequently created a stable line of neural stem cells (NSCs). These NSCs were then used to create neurons as an appropriate disease model. NPC1 neurons display a premature cell death phenotype, and gene expression analysis of these cells suggests dysfunction of important signaling pathways, including calcium and WNT. The clear readout from these cells makes them ideal candidates for high-throughput screening and will be a valuable tool to better understand the development of NPC1 in neural cells, as well as to develop better therapeutic options for NPC1. 2. Clinical trial for HP-beta-CD. I have been working with a collaborative model between the NIH, academic scientists, nonprofit organizations, and pharmaceutical and biotechnology companies to develop small molecule treatments for NPC. We published a paper describing, the development of 2-hydroxypropyl-&#946;-cyclodextrin (HP-&#946;-CD) for the treatment of Niemann-Pick disease type C1 (NPC1). The collaborative drug development program has been established between TRND, public and private partners that has completed the pre-clinical development of HP-&#946;-CD through IND filing, licensing to a company and clinical trials are underway. 3. New Therapies. Treatments currently being tested may have associated complications (cyclodextrin requires continuous delivery and may be associated with hearing loss) or only be effective in subsets of patients (HDACi may only be effective on those with mutations that affect folding/trafficking). Thus new therapies for patients with NPC need to be developed. While gene therapy for NPC was previously considered as a questionable intervention due to limitations of the field, alternative AAV serotype vectors that can transduce neurons after either systemic or local delivery have mandated a critical reappraisal of gene therapy as a possible treatment for NPC, especially since vectors in this class have proven effective in treating neurodegenerative disorders. As a critical first step that would help enable the development of a new class of gene therapy for patients, we are assessing adeno-associated virus (AAV) gene therapy as a novel therapy for NPC using a well studied murine model. We are comparing the effectiveness of systemic and intracranial injections of AAV serotypes 9 and rh10 vectors, configured to express the endogenous human NPC1. Preliminary results are promising and a patent has been filed to further develop this approach for eventual assessment in clinical trials.